1. Field of the Invention
The present invention concerns the quantitative determination of Thyroid Stimulating Hormone (hereinafter "TSH") in aqueous solutions. More particularly, the present invention provides a method and a kit for the determination of the concentration of TSH in body liquids, particularly in serum, by radioimmunoassay techniques.
TSH is a glycoprotein hormone secreted by the anterior pituitary gland and is the principal regulator of thyroid gland activity. This glycoprotein has a molecular weight of 28,000 and can be dissociated into two dissimilar sub-units of equal size, commonly designated as .alpha. and .beta.-types, which are linked to each other by non-covalent bonds and which differ from each other in their amino acid composition and sequence and in their carbohydrate composition. The subunit is similar in its amino acid composition to the .alpha. sub-units of luteinizing hormone, follicle-stimulating hormone and human chorionic gonadotropin, whereas the .beta. sub-unit of TSH is of a distinct character and is responsible for the biological and immunological specifity of this hormone. The primary functions of TSH are to stimulate thyroidal iodide metabolism and thyroid hormone synthesis and release by the thyroid gland. TSH also stimulates several other metabolic processes in the thyroid gland, including cyclic AMP generation, glucose oxidation, oxygen consumption and the synthesis of proteins and phospholipids.
TSH assay is an important diagnostic tool for diagnosing and monitoring the treatment of various disturbances of the thyroid gland function, e.g., primary hyperthyroidism. Owing to the inadequate sensitivity of current TSH radioimmunoassay methods, this assay cannot be used for the determination of lower-than-normal serum TSH levels, such as occur in hyperthyroidism.
2. Description of the Prior Art
Practically all the known methods for the determination of TSH in serum utilize the so-called "double antibody" radioimmunoassay technique with .sup.125 I-labelled TSH. Radioimmunoassay methods, in general, are based on the competition between a specific native antigen, the amount of which is to be determined in a sample, and a known amount of the same antigen in radioactively labelled form, for a limited number of available binding sites on an antibody which is specific towards the antigen under assay. Thus, in a system consisting of an unknown amount of unlabelled native antigen, a known amount of a radioactively labelled antigen and a limited known amount of antibody, the greater the concentration of unlabelled native antigen from the sample, the less the labelled antigen will be bound by the antibody. By separating the antigen-antibody complex (the "bound" species) from the remaining free antigen (both labelled and unlabelled--the "free" species) and measuring the radioactivity in one and/or the other fraction, it becomes possible to establish an assay system for measuring the unknown level of unlabelled antigen in the patient's sample. As a rule, a standard calibration curve for the specific system is established by a series of assays of standard samples with varying known amounts of unlabelled antigen and this curve is then used to determine an unknown concentration of the antigen in a sample.
There are many procedures for separating the antigen-antibody complex from the free unbound antigen, amongst which chromatoelectrophoresis, ascending paper-wick chromatography, precipitation by salts, organic materials or solvents, selective adsorption on various so-called "immunsorbents," either in suspension or on chromatographic columns, and ion exchange techniques may be mentioned as examples of such procedures. U.S. patent application Ser. No. 852,105, filed Nov. 16, 1977 and assigned to the instant assignee, is incorporated herein by reference and describes in more detail such prior art techniques for separating the bound- and free-species of the labelled antigen.
The separation of the antigen-antibody complex from the free unbound antigen is particularly difficult where the antigen is a molecule of comparatively high molecular weight (as contrasted to the relatively small and simple haptens), approaching that of the protein molecule of the antibody. Examples of such high molecular weight antigens are the various peptide hormones including the glycoprotein TSH with which the present invention is concerned. One of the separation procedures used in known radioimmunoassay methods of peptide hormones an in most, if not all, TSH radioimmunoassay tests, is the "double antibody" technique. By this technique the primary antigen-antibody complex is precipitated from its solutions as a secondary complex formed with a second antibody that was raised against the first antibody. For example, if the first antibody was raised in a rabbit against a specific antigen, then the second antibody may be an antiserum raised in goats and is specific against the first antibody or, less specifically, against rabbit gammaglobulin. This method is applicable to practically any radioimmunoassay test and results in complete separation of the antigen-antibody complex from the free native antigen, but it has the disadvantages of being comparatively costly and complicated [e.g., an additional incubation period is necessary for the formation of the antigen-(first antibody)-(second antibody) complex].
It is the object of the present invention to provide a simple, inexpensive and reliable method for the quantitative determination of TSH in aqueous solutions, particularly in serum samples.